Interleukin-5 specific recombinant antibodies

ABSTRACT

An effective anti-IL-5 recombinant antibody molecule comprising heavy and/or light chain antigen-binding residues from a donor antibody.

[0001] The present invention relates to a recombinant antibody molecule(RAM), and especially a humanized antibody molecule (HAM) havingspecificity for human interleukin-5 (hIL-5), the nucleic acids whichencode the heavy and light chain variable domains of said recombinantantibody, a process for producing said antibody using recombinant DNAtechnology and the therapeutic use of the recombinant antibody.

[0002] In the present application, the term “recombinant antibodymolecule” (RAM) is used to describe an antibody produced by a processinvolving the use of recombinant DNA technology. The term “humanizedantibody molecule” (HAM) is used to describe a molecule being derivedfrom a human immunoglobulin. The antigen binding site may compriseeither complete variable domains fused onto constant domains or one ofmore complementary determining regions (CDRs) grafted onto appropriateframework regions in the variable domain. The abbreviation “MAb” is usedto indicate a monoclonal antibody.

[0003] The term “recombinant antibody molecule” includes not onlycomplete immunoglobulin molecules but also any antigen bindingimmunoglobulin fragments, such as Fv, Fab and F(ab′)₂ fragments, and anyderivatives thereof, such as single chain Fv fragments.

[0004] Natural immunoglobulins have been used in assay, diagnosis and,to a limited extent, therapy. The use of immunoglobulins in therapy hasbeen hindered as most antibodies of potential use as therapeutic agentsare MAbs produced by fusions of a rodent spleen cells with rodentmyeloma cells. These MAbs are therefore essentially rodent proteins. Theuse of these MAbs as therapeutic agents in human can give rise to anundesirable immune response termed the HAMA (Human Anti-mouse Antibody)response. The use of rodent MAbs as therapeutic agents in humans isinherently limited by the fact that the human subject will mount animmunological response to the MAb which would either remove it entirelyor at least reduce its effectiveness.

[0005] A number of techniques to reduce the antigenic characteristics ofsuch non-human MAbs have been developed. These techniques generallyinvolve the use of recombinant DNA technology to manipulate DNAsequences encoding the polypeptide chains of the antibody molecule.These methods are generally termed “humanization” techniques.

[0006] Early methods for humanizing MAbs involved the production ofchimeric antibodies in which an antigen binding site comprising thecomplete variable domains of one antibody are fused to constant domainsderived from another antibody. Methods for carrying out suchchimerisation procedures are described in EP 0120694 (Celltech Limited)and EP 0125023 (Genentech Inc. and City of Hope). Humanized chimericantibodies, however, still contain a significant portion of non-humanamino acid sequences, and can still elicit some HAMA response,particularly if administered over a prolonged period [Begent et al., Br.J. Cancer, 62, 487 (1990)].

[0007] An alternative approach, described in EP-A-0239400 (Winter),involves the grafting of the complementarity determining region (CDRS)of a mouse MAb on to framework regions of the variable domains of ahuman immunoglobulin using recombinant DNA techniques. There are threeCDRs (CDR1, CDR2 and CDR3) in each of the heavy and light chain variabledomains. Such CDR-grafted humanized antibodies are much less likely togive rise to a HAMA response than humanized chimeric antibodies in viewof the much lower proportion of non-human amino acid sequences whichthey contain. In Riechmann et al. [Nature, 332 323-324 (1988)] it wasfound that the transfer of the CDRs alone, as defined by Kabat[Sequences or Proteins of Immunological Interest, US Department ofHealth and Human Services, NIH, USA (1987)], was not sufficient toprovide satisfactory antigen binding activity in the CDR-graftedproduct. Riechmann et al. found that it was necessary to convert anumber of residues outside the CDRS, in particular in the loop adjacentCDR1. However, the binding affinity of the best CDR-grafted antibodiesobtained was still significantly less than that of the original MAb.

[0008] In WO 91/09967, Adair et al. described CDR-grafted antibody heavyand light chains, and determined a hierarchy of donor residues.

[0009] In WO 93/16184, Chou et al. described the design, cloning andexpression of humanized monoclonal antibodies against humaninterleukin-5. A method for selecting human antibody sequences to beused as human frameworks for humanization of an animal antibody issuggested, comprising the steps of comparing human variable domainsequences with the variable domain sequences of the animal MAb that isto be humanized for percentage identities, sequence ambiguities andsimilar PIN-region spacing. PIN-region spacing is defined as the numberof residues between the cysteine residues forming the intra domaindisulfide bridges. The human antibody having the best combination ofthese features is selected. A method for determining which variabledomain residues of an animal MAb which should be selected forhumanization is also suggested, comprising determining potential minimumresidues (residues which comprise CDR structural loops and the residuesrequired to support and/or orientate the CDR structural loops) andmaximum residues (residues which comprise Kabat CDRs, CDR structuralloops, residues required to support and/or orientate the CDR structuralloops and residues which fall within about 10 Å of a CDR structural loopand possess a water solvent accessible surface of about 5 Å² or greater)of the animal monoclonal antibody. Furthermore, computer modelling isperformed on all possible recombinant antibodies, comprising the humanantibody framework sequence into which minimum and maximum residues havebeen inserted. The minimum or maximum residues are selected based on thecombination which produces a recombinant antibody having acomputer-model structure closest to that of the animal monoclonalantibody. The humanized anti-IL-5 antibody obtained appears to have losta substantial amount of its affinity for the hIL-5 molecule.

[0010] It is an aim of the present invention to provide a humanizedantibody molecule having improved affinity for the hIL-5 molecule.

[0011] Accordingly the present invention provides a RAM having affinityfor human IL-5 and comprising antigen binding regions derived from heavyand/or light chain variable domains of a donor antibody having affinityfor human IL-5, the RAM having a binding affinity similar to that of thedonor antibody.

[0012] The RAM of invention may comprise antigen binding regions fromany suitable donor anti-IL-5 antibody. Typically the donor anti-IL-5antibody is a rodent MAb. Preferably the donor antibody is MAb 39D10.

[0013] The variable domains of the heavy and light chains of MAb 39D10are hereinafter specifically described with reference to FIGS. 1 and 2.

[0014] According to one preferred aspect of the invention, the RAM ofthe present invention is an anti-IL-5 antibody molecule having affinityfor the human IL-5 antigen comprising a composite heavy chain and acomplementary light chain, said composite heavy chain having a variabledomain comprising predominantly acceptor antibody heavy chain frameworkresidues and donor antibody heavy chain antigen-binding residues, saiddonor antibody having affinity for human IL-5, wherein said compositeheavy chain comprises donor residues at least at positions 31-35, 50-65and 95-102 (according to the Kabat numbering system) [Kabat et al.,Sequences of Proteins of Immunological Interest, Vol I, Fifth Edition,1991, US Department of Health and Human Services, National Institute ofHealth].

[0015] Preferably, the composite heavy chain framework additionallycomprises donor residues at positions 23, 24, 27-30, 37, 49, 73 and76-78 or 24, 27-30, 37, 49, 73, 76 and 78.

[0016] According to a second preferred aspect of the present invention,there is provided an anti-IL-5 antibody molecule having affinity for ahuman IL-5 antigen comprising a composite light chain and acomplementary heavy chain, said composite light chain having a variabledomain comprising predominantly acceptor antibody light chain frameworkresidues and donor antibody light chain antigen-binding residues, saiddonor antibody having affinity for human IL-5, wherein said compositelight chain comprises donor residues at least at positions 24-34, 50-56and 89-97 (according to the Kabat numbering system).

[0017] Preferably, the composite light chain framework additionallycomprises donor residues at positions 22, 68 and 71 or at positions 68and 71.

[0018] According to a third preferred aspect of the present invention,there is provided an anti-IL-5 antibody molecule having affinity for ahuman IL-5 antigen comprising a composite heavy chain according to thefirst aspect of the invention and a composite light chain according tothe second aspect of the invention.

[0019] Preferably, each RAM of the invention has an affinity constantfor human IL-5 of greater than 10⁻⁹M.

[0020] It will be appreciated that the invention is widely applicable tothe production of anti-IL-5 RAMs in general. Thus, the donor antibodymay be any anti-IL-5 antibody derived from any animal. The acceptorantibody may be derived from an animal of the same species and may evenbe of the same antibody class or sub-class. More usually, however, thedonor and acceptor antibodies are derived from animals from differentspecies. Typically, the donor anti-IL-5 antibody is a non-humanantibody, such as a rodent MAb, and the acceptor antibody is a humanantibody.

[0021] Any appropriate acceptor variable framework sequence may be usedhaving regard to class or type of the donor antibody from which theantigen binding regions are derived. Preferably, the type of acceptorframework used is of the same or similar class or type as that of thedonor antibody. Conveniently, the framework chosen has the most homologyto the donor antibody. Preferably, the human group III gamma germ lineframeworks are used for the composite heavy chain and the human group Ikappa germ line frameworks are used for the composite light chains.

[0022] The constant region domains of the RAMs of the invention may beselected having regard to the proposed functions of the antibody, inparticular the effector functions which may be required. For example,the constant region domains may be human IgA, IgE, IgG or IgM domains.In particular, IgG human constant region domains may be used, especiallyof the IgG1 and the IgG3 isotype, when the humanized antibody moleculeis intended for therapeutic uses, and antibody effector functions arerequired. Alternatively, IgG2 and IqG4 isotypes may be used where thehumanized antibody molecule is intended for therapeutic purposes andantibody effector functions are not required, e.g. for specificallybinding to and neutralizing the biological activity of human IL-5.Modified human constant region domains may also be used in which one ormore amino acid residues have been altered or deleted to change aparticular effector function. Preferably, the constant region domains ofthe RAMs are human IgG4.

[0023] The residue designations given above and elsewhere in the presentapplication are numbered according to the Kabat numbering [Kabat et al.,Sequences of Proteins of Immunological Interest, Vol I, Fifth Edition,1991, US Department of Health and Human Services, National Institute ofHealth]. Thus, the residue designations do not always corresponddirectly with a linear numbering of the amino acid residues. The actuallinear amino acid sequence may contain fewer or additional amino acidsthan in the Kabat numbering, corresponding to a shortening of, orinsertion into, the basic variable domain structure.

[0024] Also the anti-IL-5 antibody molecules of the present inventionmay have attached to them effector or reporter molecules. Alternatively,the procedures of recombinant DNA technology may be used to produceimmunoglobulin molecules in which the Fc fragment or CH3 domain of acomplete immunoglobulin has been replaced by, or has been attachedthereto by peptide linkage, a functional non-immunoglobulin protein,such as an enzyme, cytokine, growth factor or toxin molecule.

[0025] Thus, the remainder of the antibody molecules need not compriseonly sequences from immunoglobulins. For instance, a gene may beconstructed in which a DNA sequence encoding part of a humanimmunoglobulin chain is fused to a DNA sequence encoding the amino acidsequence of a polypeptide effector or reporter molecule.

[0026] Further aspects of the invention include DNA sequences coding forthe composite heavy chain and the composite light chain. The cloning andexpression vectors containing the DNA sequences, host calls transformedwith the DNA sequences and the processes for producing the antibodymolecules comprising expressing the DNA sequences in the transformedhost cells are also further aspects of the invention.

[0027] The general methods by which vectors may be constructed,transfection methods and culture methods are well known in the art andform no part of the invention.

[0028] The DNA sequences which encode the anti-IL-5 donor amino acidsequences may be obtained by methods well known in the art (see, forexample, International Patent Application No. WO 93/16184). For example,the anti-IL-5 coding sequences may be obtained by genomic cloning orcDNA cloning from suitable hybridoma cell lines, e.g. the 39D10 cellline. Positive clones may be screened using appropriate probes for theheavy and light chains required. Also PCR cloning may be used.

[0029] The DNA coding for acceptor amino acid sequences may be obtainedin any appropriate way. For example, DNA sequences coding for preferredhuman acceptor frameworks such as human group I light chains and humangroup III heavy chains, are widely available to workers in the art.

[0030] The standard techniques of molecular biology may be used toprepare the desired DNA sequences. The sequences may be synthesisedcompletely or in part using oligonucleotide synthesis techniques.Site-directed mutagenesis and polymerase chain reaction (PCR) techniquesmay be used as appropriate. For example, oligonucleotide directedsynthesis as described by Jones et al. [Nature, 321, 522 (1986)] may beused. Also oligonucleotide directed mutagenesis of a preexistingvariable region as, for example, described by Verhoeyen et al. [Science,239, 1534-1536 (1988)] may be used. Also enzymatic filling in of gappedoligonucleotides using T4 DNA polymerase as, for example, described byQueen et al. [Proc. Natl. Acad. Sci. USA, 86, 10029-10033 (1989) and WO90/07861] may be used.

[0031] Any suitable host cell and vector system may be used for theexpression of DNA sequences coding for the RAM. Preferably, eucaryotic,e.g. mammalian, host cell expression systems are used. In particular,suitable mammalian host cells include CHO cells and myeloma or hybridomacell lines.

[0032] Thus, according to a further aspect of the present invention aprocess for producing an anti-IL-5 RAM is provided comprising:

[0033] (a) producing in a first expression vector a first operon havinga DNA sequence which encodes a composite heavy chain, as definedaccording to the first preferred aspect of the invention;

[0034] (b) optionally producing in the first or a second expressionvector a second operon having a DNA sequence which encodes acomplementary light chain, which may be a composite light chain asdefined according to the second preferred aspect of the invention;

[0035] (c) transfecting a host cell with the or each vector; and

[0036] (d) culturing a transfected cell line to produce the RAM.

[0037] Alternatively, the process may involve the use of sequencesencoding a composite light chain and a complementary heavy chain.

[0038] For the production of RAMs comprising both heavy and lightchains, the cell lines may be transfected with two vectors. The firstvector may contain an operon encoding a composite or complementary heavychain and the second vector may contain an operon encoding acomplementary or composite light chain. Preferably, the vectors areidentical except insofar as the coding sequences and selectable markersare concerned so as to ensure as far as possible that each polypeptidechain is equally expressed. In a preferred alternative, a single vectormay be used, the vector including the sequences encoding both the heavychain and the light chain.

[0039] The DNA in the coding sequences for the heavy and light chainsmay comprise cDNA or genomic DNA or both.

[0040] The present invention also includes therapeutic and diagnosticcompositions comprising the RAMS and uses of such compositions intherapy and diagnosis.

[0041] Accordingly, in a further aspect the invention provides atherapeutic or diagnostic composition comprising a RAM according toprevious aspects of the invention in combination with a pharmaceuticallyacceptable excipient, diluent or carrier.

[0042] These compositions can be prepared using the RAMs of the presentinvention, for instance as whole antibodies, single chain Fv fragmentsor antibody fragments, such as Fab or Fv fragments. Such compositionshave IL-5 blocking or antagonistic effects and can be used to suppressIL-5 activity.

[0043] The compositions according to the invention may be formulated inaccordance with conventional practice for administration by any suitableroute, and may generally be in a liquid form [e.g. a solution of the RAMin a sterile physiologically acceptable buffer] for administration byfor example an intravenous, intraperitoneal or intramuscular route; inspray form, for example for administration by a nasal or buccal route;or in a form suitable for implantation.

[0044] The invention also provides a method of therapy or diagnosiscomprising administering an effective amount, preferably 0.1 to 10 mg/kgbody weight, of a RAM according to previous aspects of the invention toa human or animal subject. The exact dosage and total dose will varyaccording to the intended use of the RAM and on the age and condition ofthe patient to be treated. The RAM may be administered as a single done,or in a continuous manner over a period of time. Doses may be repeatedas appropriate.

[0045] The RAM according to previous aspects of the invention may beused for any of the therapeutic uses for which anti-IL-5 antibodies,e.g. 39D10, have been used or may be used in the future.

[0046] IL-5 is a primary activator of eosinophils, and blocking thefunction of this cytokine with antibodies has been shown to prevent orreduce eosinophilia which is associated with certain allergic diseases.Thus the RAM according to the invention may be used for this purpose,and in particular may be of use in the treatment of asthma, where it maybe expected to prevent the accumulation and activation of eosinophils inasthmatic lungs, thereby reducing bronchial inflammation and airwaynarrowing. For use in the treatment of asthma the RAM according to theinvention may advantageously be a single chain Fv fragment, formulatedas a spray, for administration for example via the nasal route.

[0047] A preferred protocol for obtaining an anti-IL5 antibody moleculein accordance with the present invention is set out below. This protocolis given without prejudice to the generality of the invention ashereinbefore described and defined.

[0048] The 39D10 rat monoclonal antibody raised against human IL-5 isused as the donor antibody. The variable domains of the heavy and lightchains of 39D10 have previously been cloned (WO 93/16184) and thenucleotide and predicted amino acid sequences of these domains are shownin FIGS. 1 and 2. The appropriate acceptor heavy and light chainvariable domains must be determined and the amino acid sequence known.The RAM is then designed starting from the basis of the acceptorsequence.

[0049] 1. The CDRs

[0050] At a first step, donor residues are substituted for acceptorresidues in the CDRS. For this purpose, the CDRs are preferably definedas follows: heavy chain: CDR1: residues 31-35 CDR2: residues 50-65 CDR3:residues 95-102 light chain: CDR1: residues 24-34 CDR2: residues 50 to56 CDR3: residues 89 to 97

[0051] The positions at which donor residues are to be substituted foracceptor residues in the framework are then chosen as follows, first ofall with respect to the heavy chain and subsequently with respect to thelight chain.

[0052] 2. Heavy Chain

[0053] 2.1 Donor residues are used either at all of positions 24, 27 to30, 37, 49, 73, 76 and 78 or at all of positions 23, 24, 27 to 30, 37,49, 73 and 76 to 78 of the heavy chain.

[0054] 3. Light Chain

[0055] 3.1 Donor residues are used either at all of positions 22, 68 and71 or at all of positions 68 and 71.

[0056] The present invention relates to a recombinant anti-IL-5 antibodymolecule having a binding affinity substantially equal to that of thedonor antibody. The present invention is now described, by way ofexample only, with reference to the accompanying drawings, in which:

[0057]FIG. 1 shows the nucleotide and amino acid sequence of the 39D10heavy chain;

[0058]FIG. 2 shows the nucleotide and amino acid sequence of the 39D10light chain;

[0059]FIG. 3 shows the alignment of the 39D10 heavy chain variabledomain framework regions with the heavy chain variable domain frameworkregions of the consensus sequence of the human group III heavy chains;

[0060]FIG. 4 shows the alignment of the 39D10 light chain variabledomain framework regions with the light chain variable domain frameworkregions of the consensus sequence of the human group I light chains;

[0061]FIG. 5 shows the nucleotide and amino acid sequence of the CDRgrafted anti-IL-5 light chain CTIL-5-gL6;

[0062]FIG. 6 shows the nucleotide and amino acid sequence of the CDRgrafted anti-IL-5 heavy chain CTIL-5-10gH;

[0063]FIG. 7 shows a map of plasmid pHR14;

[0064]FIG. 8 shows a map of plasmid pMR15.1;

[0065]FIG. 9 shows the affinity constants and association anddisassociation rates of a chimeric 39D10 antibody and theCTIL-5-10gH\-gL6 antibody;

[0066]FIG. 10 shows a graph of the neutralisation of IL-5 in the TF1assay by a panel of antibodies;

[0067]FIG. 11 shows the results of a competition assay for rat 39D10, achimeric 39D10 antibody and the CTIL-5-10gH/qL6 antibody; and

[0068]FIG. 12 shows the effect of CTIL-5-10gH/gL6 on monkeyeosinophilia.

EXAMPLE

[0069] 1. Material and Methods

[0070] 39D10 is a rat monoclonal antibody raised against human IL-5. Thegenes for the variable domains of the heavy and light chains of 39D10have previously been cloned (WO 93/16184) and the nucleotide andpredicted amino acid sequences of these domains are shown in FIGS. 1 and2. Because of the strategy used in the cloning of the variable domain ofthe 39D10 heavy chain, the first five amino acids of the frameworkregions are unknown. However, a heavy chain was available whichcontained the leader sequence and the first five amino acids offramework 1 from the antibody YTH 34.5HL, Riechmann et al., [Nature, 332323-327 (1988)].

[0071] 2. Molecular Biology Procedures

[0072] The molecular biology procedures used were as described inManiatis et al. [Molecular Cloning: A Laboratory Manual, Second Edition,Vols 1 to 3, Cold Spring Harbor Laboratory Press (1989)].

[0073] 3. Construction of Recombinant Heavy and Light Chain Genes

[0074] Heavy Chain

[0075] A heavy chain Vh region was generated by PCR using theoligonucleotides R3601 and R2155. The sequences of these are:

[0076] R3601 5′GCGCGCAAGCTTGCCGCCACCATGAAG(A, T)TGTGGTTAAACTGGGTTTT3′

[0077] R2155 5′GCAGATGGGCCCTTCGTTGAGGCTG(A, C) (A, G)GAGAC(G, T,A)GTGA3′

[0078] The reaction mixture (100 μl) contained 10 mM Tris-HCl pH 8.3,1.5 mM MgCl, 50 mM KCl, 0.01% w/v gelatin, 0.25 mM of eachdeoxyribonucleoside triphosphate, 0.1 μg 39D10 heavy chain DNA, 6 pmolesof R3601 and R2155 and 0.25 units Taq polymerase. The reaction mixturewas heated at 94° C. for 5 minutes and then cycled through 94° C. for 1minute, 55° C. for 1 minute and 72° C. for 1 minute. After 30 cycles,the reaction was extracted with an equal volume of phenol/chloroform(1:1 v/v), then with chloroform before being precipitated by theaddition of 2.5 volumes of ethanol. The PCR product was dissolved in theappropriate buffer, digested with HindIII and ApaI, purified on anagarose gel and ligated into the vector pMR14 (FIG. 7) which had alsobeen digested with HindIII and ApaI. Following transformation into E.coli LM1035, colonies were grown overnight and plasmid DNA analysed forVh inserts. The nucleotide sequence of the vh region in plasmid,pARH1217, is shown in FIG. 1.

[0079] Light Chain

[0080] A Vl light chain gene was generated from the original Vl, asdescribed in WO 93/16184, clone by PCR with the oligonucleotides R3585and R3597. The sequences of these are:

[0081] R3585 5′GGACTGTTCGAAGCCGCCACCATGAGTGTGCTCACTCAGGTCCT3′

[0082] R3597 5′GGATACAGTTGGTGCAGCATCCGTACGTTT3′

[0083] PCR was carried out as described above. The PCR product wasdigested with the enzymes BstBI and SplI and, after purification,ligated into pMR15.1 (FIG. 8) that had previously been digested with thesame enzymes.

[0084] A colony was identified, after transformation of E. coli LM1035,that contained a plasmid (pARH1215) with a Vl insert. The nucleotidesequence of the Vl insert is shown in FIG. 2.

[0085] CDR Grafting of 39D10

[0086] Light Chain

[0087] In order to decide on the most appropriate human acceptorframeworks for the CDR loops of 39D10, the amino acid sequence offrameworks 1-3 of 39D10 were compared with those of known human kappalight chains. 39D10 was found to be most homologous to human group Ilight chains. Based on this, it was decided to use the human group Igerm line frameworks for the CDR grafting. The homologies between thesesequences are shown in FIG. 3. Also shown is the homology between theframework 4 regions of 39D10 and the consensus sequence of known humangroup I light chains. The residues in 39D10 that differ from the humanconsensus sequence are underlined. The contribution that these residuesmight make to antigen binding was analysed and two genes wereconstructed for the CDR grafted light chain. These were CTIL-5-gL5 andCTIL-5-gL6 in which, as well as the CDR residues, either residues 22, 68and 71 or residues 68 and 71 were also from 39D10 respectively. Thenucleotide and amino acid sequences of CTIL-5-gL6 are shown in FIG. 5.

[0088] Heavy Chain

[0089] CDR grafting of the 39D10 heavy chain was carried out asdescribed for the light chain. The framework regions of 39D10 were foundto be most homologous to those of human group III antibodies and,consequently, the consensus sequence of the frameworks of the humangroup III germ line genes was used to accept the CDRs of the 39D10 heavychain. As before, the consensus sequence for human group III framework 4regions was also chosen. A comparison of these sequences is shown inFIG. 4 with the residues in 39D10 that differ from the human consensussequence underlined.

[0090] Analysis of the framework residues in 39D10 that might influenceantigen binding was carried out and, based on this, two genes,CTIL-5-9gH and CTIL-5-10gH, were constructed in which either residues23, 24, 27 to 30, 37, 49, 73 and 76 to 78 or residues 24, 27-30, 37, 49,73, 76 and 78 respectively were from 39D10. The nucleotide and aminoacid sequences of CTIL-5-10gh is shown in FIG. 6.

[0091] Expression and Bioactivity of Anti-IL-5 Antibodies

[0092] Chimeric (rat/human) and CDR grafted 39D10 were produced forbiological evaluation by transient expression of the heavy and lightchain pairs after co-transfection into Chinese Hamster Ovary (CHO) cellsusing calcium phosphate precipitation.

[0093] On the day prior to transfection, semi-confluent flasks ofCHO-L761h cells (Cockett et al., Nucl. Acids. Res., 19, 319-325, 1991)were trypsinised, the cells counted and T75 flasks set up each with 10⁷cells. On the next day, the culture medium was changed 3 hours beforetransfection. For transfection, the calcium phosphate precipitate wasprepared by mixing 1.25 ml of 0.25M CaCl₂ containing 50 μg of each ofheavy and light chain expression vectors with 1.25 ml of 2×HBS (16.36 gNaCl, 11.9 gm HEPES and 0.4 g Na ₂HPO₆ in 1 litre water with the pHadjusted to 7.1 with NaOH) and adding immediately into the medium of thecells. After 3 hours at 37° C. in a CO₂ incubator, the medium andprecipitate were removed and the cells shocked by the addition of 15 ml15% glycerol in phosphate buffered saline (PBS) for 1 minute. Theglycerol was removed, the cells washed once with PBS and incubated for48-96 hours in 25 ml medium containing 10 mM sodium butyrate. Antibodywas purified from the culture medium by binding to and elution fromprotein A-Sepharose. Antibody concentration was determined using a humanIg ELISA (see below).

[0094] ELISA

[0095] Antibody expression was assessed by transfecting pairs of heavyand light chain genes into CHO cells and, after three days incubation,measuring the amount of antibody accumulating in the culture medium byELISA.

[0096] For the ELISA, Nunc ELISA plates were coated overnight at 4° C.with a F(ab′)₂ fragment of a polyclonal goat anti-human Fc fragmentspecific antibody (Jackson Immunoresearch, code 109-006-098) at 5 μg/mlin coating buffer (15 mM sodium carbonate, 35 mM sodium hydrogencarbonate, pH6.9). Uncoated antibody was removed by washing 5 times withdistilled water. Samples and purified standards to be quantitated werediluted to approximately 1 μg/ml in conjugate buffer (0.1M Tris-HClpH7.0, 0.1M NaCl, 0.2% v/v Tween 20, 0.2% w/v Hammersten casein). Thesamples were titrated in the microtitre wells in 2-fold dilutions togive a final volume of 0.1 ml in each well and the plates were incubatedat room temperature for 1 hour with shaking. After the first incubationstep, the plates were washed 10 times with distilled water and thenincubated for 1 hour as before with 0.1 ml of a mouse monoclonalanti-human kappa (clone GD12) peroxidase conjugated antibody (TheBinding Site, code MP135) at a dilution of 1 in 700 in conjugate buffer.The plate was washed again and substrate solution (0.1 ml) added to eachwell. Substrate solution contained 150 μl N,N,N,N-tetramethylbenzidine(10 mg/ml in DMSO), 150 μl hydrogen peroxide (30% solution) in 10 ml0.1M sodium acetate/sodium citrate, pH6.0. The plate was developed for5-10 minutes until the absorbence at 630 nm was approximately 1.0 forthe top standard. Absorbence at 630 nm was measured using a plate readerand the concentration of the sample determined by comparing thetitration curves with those of the standard.

[0097] Determination of Affinity Constants for Anti-IL-5 Antibodies

[0098] Affinities of the chimeric and CDR grafted anti-IL-5 antibodieswere determined using Biospecific Interaction Analysis (BIA). Antibodieswere produced in CHO cells by transfection of combinations of heavy andlight chain genes and purified from culture supernatants on Protein ASepharose. For affinity measurements, a polyclonal anti-human Fcantibody was bound to the Pharmacia Biosensor chip (12150 relativeresponse units, RU) and used to capture anti-IL-5 which was passed overthe chip at 5 μg/ml in 10 mM HEPES, 0.15M NaCl, 3.4 mM EDTA, pH7.4. Theamount of anti-IL-5 captured for each run was approximately 1600 RU.Recombinant human IL-5 was then passed over the Sensorchip at variousconcentrations (0.6 to 5 μg/ml) in the above buffer. The Sensorchip wascleaned after each run with 100 mM HCl and 100 mM orthophosphoric acidto remove bound IL-5 and antibody. The sensorgrams generated wereanalysed using the kinetics software available with the BIAcore machine.

[0099] Values for the affinity constants and association anddissociation rates of two antibodies, chimeric 39D10 andCTIL-5-10gH/-gL6, were determined. The results are shown in FIG. 9. Itcan be seen that chimeric 39D10 has an extremely high affinity for humanIL-5 and that this value has been reproduced in CTIL-5-10gH/-gL6.

[0100] Activity of Anti-IL-5 Antibodies in in vitro Bioassay

[0101] The activities of various CDR grafted antibodies were comparedwith that of chimeric 39D10 in an in vitro bioassay using TF1 cells. TF1is an erythroleukemic cell line that requires GM-CSF for growth. GM-CSFcan be replaced by IL-5 but in this instance the cells only survive anddo not proliferate. However the dependence on IL-5 for survival meansthat TF1 cells can be used in a bioassay to compare the activities ofvarious anti-IL-5 antibodies.

[0102] Neutralisation by anti-IL-5 antibodies was measured using aconstant amount of IL-5 (2 ng/ml) and variable amounts of antibodyincubated with 5×10⁴ cells per well in 96 flat bottomed plates for 3days. For the last 4 hours, cells are cultured in the presence of 500μg/ml Thiazolyl blue (MIT). This dye is converted into an insolublepurple form by mitochondrial enzymes in viable cells. The insolublematerial was dissolved by incubating overnight after addition of 100 μlof 50% dimethyl formamide, 20% SDS pH4.7 and the amount of dye taken updetermined spectrophotometrically. The levels of bioactive IL-5remaining in the presence of the antibodies is extrapolated from astandard curve relating dye uptake to IL-5 concentration.

[0103] The activities of various combination of heavy and light chainswere evaluated using the TF1 bioassay. The results are shown in FIG. 10.It can be seen that all combinations of CDR grafted heavy and lightchains produce antibodies that are equipotent with chimeric 39D10. Theseresults indicate that neither residue 22 in the light chain nor residues23 or 78 in the heavy chain are required to be 39D10 specific foroptimal binding. The combination with the fewer 39D10 specific residuesis therefore CTIL-5-10 gH/-gL6.

[0104] Activity of Anti-IL-5 Antibodies in Competition Assays

[0105] Recombinant human IL-5 was diluted to 1 μg/ml in phosphatebuffered saline (PBS) and 100 μl aliquots added to microtitre plates(Costar Amine Binding plates) and incubated overnight at 4°C. Plateswere washed three times with PBS containing 0.5% Tween 20 and anyremaining active sites blocked with 2% bovine serum albumin (BSA) in PBSfor 30 minutes. The plates were then aspirated and tapped dry. Tocompare the relative binding activity of the parent rat antibody (39D10)with chimeric and grafted antibodies, serial dilutions were prepared ofeach anti-IL-5 antibody in PBS/1% BSA and 50 μl added to duplicate wellsfollowed immediately by 50 μl 39D10-biotin conjugate at 0.125 μg/ml. Theassay was incubated for 2 hours at room temperature with agitation andthen washed twice with PBS. Horseradish-peroxidase conjugated tostreptavidin (1 μg/ml) was added to all wells and incubated for afurther 30 minutes. Plates were washed four times and 100 μl tetramethylbenzidine (TMB) substrate added. Colour development was read at 630 nm(reference 490 nm) and OD (630-490) was plotted against log (10)antibody concentration.

[0106] When the activities of rat 39D10, chimeric 39D10 andCTIL-5-10gH/gL6 were compared in the above competition assay, theresults shown in FIG. 11 were obtained. All three antibodies competedequally well with biotinylated-39D10 for binding to IL-5, indicatingthat the CDR loops of 39D10 had been successfully transferred to thehuman frameworks.

[0107] Effect of Anti-IL-5 Antibody on Monkey Eosinophilia

[0108] Anti-IL-5 antibody (CTIL-5-10gH/gL6) was tested in a monkeysystem which models asthmatic conditions (see Mauser, P.J. et al., Ann.Rev. Respir. Dis., in press). When administered, one hour beforechallenge with Ascaris, to responsive monkeys, CTIL-5-10gH/gL6 inhibitslung lavage eosinophilia 75% at a dose of 0.3 mg/kg i.v. This set ofmonkeys is not hyper-responsive to histamine so the effects ofCTIL-5-10gH/gL6 on hyper-responsiveness could not be determined. Threemonths after this single dose, eosinophil accumulation in response toAscaris challenge is still inhibited 75%.

[0109] In the allergic mouse, CTIL-5-10gH/gL6 inhibits pulmonaryeosinophilia at 1 mg/kg i.p.

1 28 1 47 DNA Artificial Sequence Primer 1 gcgcgcaagc ttgccgccaccatgaagwtg tggttaaact gggtttt 47 2 37 DNA Artificial Sequence Primer 2gcagatgggc ccttcgttga ggctgmrgag acdgtga 37 3 44 DNA Artificial SequencePrimer 3 ggactgttcg aagccgccac catgagtgtg ctcactcagg tcct 44 4 30 DNAArtificial Sequence Primer 4 ggatacagtt ggtgcagcat ccgtacgttt 30 5 333DNA Rattus rattus 5 gaatctggag gaggcttggt acagccatca cagaccctgtctctcacctg cactgtctct 60 gggttatcat taaccagcaa tagtgtgaac tggattcggcagcctccagg aaagggtctg 120 gagtggatgg gactaatatg gagtaatgga gacacagattataattcagc tatcaaatcc 180 cgactgagca tcagtaggga cacctcgaag agccaggttttcttaaagat gaacagtctg 240 caaagtgaag acacagccat gtacttctgt gccagagagtactacggcta ctttgattac 300 tggggccaag gagtcatggt cacagtctcc tca 333 6 111PRT Rattus rattus 6 Glu Ser Gly Gly Gly Leu Val Gln Pro Ser Gln Thr LeuSer Leu Thr 1 5 10 15 Cys Thr Val Ser Gly Leu Ser Leu Thr Ser Asn SerVal Asn Trp Ile 20 25 30 Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Met GlyLeu Ile Trp Ser 35 40 45 Asn Gly Asp Thr Asp Tyr Asn Ser Ala Ile Lys SerArg Leu Ser Ile 50 55 60 Ser Arg Asp Thr Ser Lys Ser Gln Val Phe Leu LysMet Asn Ser Leu 65 70 75 80 Gln Ser Glu Asp Thr Ala Met Tyr Phe Cys AlaArg Glu Tyr Tyr Gly 85 90 95 Tyr Phe Asp Tyr Trp Gly Gln Gly Val Met ValThr Val Ser Ser 100 105 110 7 384 DNA Rattus rattus 7 atggctgtgcccactcagct cctggggttg ttgttgctgt ggattacaga tgccatatgt 60 gacatccagatgacacagtc tccagcttcc ctgtctgcat ctctgggaga aactatctcc 120 atcgaatgtctagcaagtga gggcatttcc agttatttag cgtggtatca gcagaagcca 180 gggaaatctcctcagctcct gatctatggt gcaaatagct tgcaaactgg ggtcccatca 240 cggttcagtggcagtggatc tgccacacaa tattctctca agatcagcag catgcaacct 300 gaagatgaaggggattattt ctgtcaacag agttacaagt ttccgaacac gtttggagct 360 gggaccaagctggaactgaa acgg 384 8 128 PRT Rattus rattus 8 Met Ala Val Pro Thr GlnLeu Leu Gly Leu Leu Leu Leu Trp Ile Thr 1 5 10 15 Asp Ala Ile Cys AspIle Gln Met Thr Gln Ser Pro Ala Ser Leu Ser 20 25 30 Ala Ser Leu Gly GluThr Ile Ser Ile Glu Cys Leu Ala Ser Glu Gly 35 40 45 Ile Ser Ser Tyr LeuAla Trp Tyr Gln Gln Lys Pro Gly Lys Ser Pro 50 55 60 Gln Leu Leu Ile TyrGly Ala Asn Ser Leu Gln Thr Gly Val Pro Ser 65 70 75 80 Arg Phe Ser GlySer Gly Ser Ala Thr Gln Tyr Ser Leu Lys Ile Ser 85 90 95 Ser Met Gln ProGlu Asp Glu Gly Asp Tyr Phe Cys Gln Gln Ser Tyr 100 105 110 Lys Phe ProAsn Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Arg 115 120 125 9 23 PRTArtificial Sequence Consensus 9 Asp Ile Gln Met Thr Gln Ser Pro Ser SerLeu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys 20 10 23PRT Rattus rattus 10 Asp Ile Gln Met Thr Gln Ser Pro Ala Ser Leu Ser AlaSer Leu Gly 1 5 10 15 Glu Thr Ile Ser Ile Glu Cys 20 11 15 PRTArtificial Sequence Consensus 11 Trp Tyr Gln Gln Lys Pro Gly Lys Ala ProLys Leu Leu Ile Tyr 1 5 10 15 12 15 PRT Rattus rattus 12 Trp Tyr Gln GlnLys Pro Gly Lys Ser Pro Gln Leu Leu Ile Tyr 1 5 10 15 13 32 PRTArtificial Sequence Consensus 13 Gly Val Pro Ser Arg Phe Ser Gly Ser GlySer Gly Thr Asp Phe Thr 1 5 10 15 Leu Thr Ile Ser Ser Leu Gln Pro GluAsp Phe Ala Thr Tyr Tyr Cys 20 25 30 14 32 PRT Rattus rattus 14 Gly ValPro Ser Arg Phe Ser Gly Ser Gly Ser Ala Thr Gln Tyr Ser 1 5 10 15 LeuLys Ile Ser Ser Met Gln Pro Glu Asp Glu Gly Asp Tyr Phe Cys 20 25 30 1511 PRT Artificial Sequence Consensus 15 Phe Gly Gln Gly Thr Lys Val GluIle Lys Arg 1 5 10 16 11 PRT Rattus rattus 16 Phe Gly Ala Gly Thr LysLeu Glu Leu Lys Arg 1 5 10 17 30 PRT Artificial Sequence Consensus 17Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 1015 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser 20 25 30 1830 PRT Rattus rattus MISC_FEATURE (1)..(5) Xaa is unknown 18 Xaa Xaa XaaXaa Xaa Glu Ser Gly Gly Gly Leu Val Gln Pro Ser Gln 1 5 10 15 Thr LeuSer Leu Thr Cys Thr Val Ser Gly Leu Ser Leu Thr 20 25 30 19 14 PRTArtificial Sequence Consensus 19 Trp Val Arg Gln Ala Pro Gly Lys Gly LeuGlu Trp Val Ser 1 5 10 20 14 PRT Rattus rattus 20 Trp Ile Arg Gln ProPro Gly Lys Gly Leu Glu Trp Met Gly 1 5 10 21 32 PRT Artificial SequenceConsensus 21 Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr LeuGln 1 5 10 15 Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr CysAla Arg 20 25 30 22 32 PRT Rattus rattus 22 Arg Leu Ser Ile Ser Arg AspThr Ser Lys Ser Gln Val Phe Leu Lys 1 5 10 15 Met Asn Ser Leu Gln SerGlu Asp Thr Ala Met Tyr Phe Cys Ala Arg 20 25 30 23 11 PRT ArtificialSequence Consensus 23 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 1 5 1024 11 PRT Rattus rattus 24 Trp Gly Gln Gly Val Met Val Thr Val Ser Ser 15 10 25 399 DNA Artificial Sequence Novel Sequence 25 ttcgaagccgccaccatgtc tgtccccacc caagtcctcg gtctcctgct gctgtggctt 60 acagatgccagatgtgacat tcaaatgacc cagagcccat ccagcctgag cgcatctgta 120 ggagaccgggtcaccatcac atgtctagca agtgagggca tctccagtta cttagcgtgg 180 taccagcagaagcccgggaa agctcctaag ctcctgatct atggtgcgaa tagcttgcag 240 actggagtaccatcaagatt cagtggctca ggatccgcta cagactacac gctcacgatc 300 tccagcctacagcctgaaga tttcgcaacg tattactgtc aacagtcgta taagttcccg 360 aacacattcggtcaaggcac caaggtcgaa gtcaaacgt 399 26 128 PRT Artificial Sequence NovelSequence 26 Met Ser Val Pro Thr Gln Val Leu Gly Leu Leu Leu Leu Trp LeuThr 1 5 10 15 Asp Ala Arg Cys Asp Ile Gln Met Thr Gln Ser Pro Ser SerLeu Ser 20 25 30 Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Leu Ala SerGlu Gly 35 40 45 Ile Ser Ser Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly LysAla Pro 50 55 60 Lys Leu Leu Ile Tyr Gly Ala Asn Ser Leu Gln Thr Gly ValPro Ser 65 70 75 80 Arg Phe Ser Gly Ser Gly Ser Ala Thr Asp Tyr Thr LeuThr Ile Ser 85 90 95 Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys GlnGln Ser Tyr 100 105 110 Lys Phe Pro Asn Thr Phe Gly Gln Gly Thr Lys ValGlu Val Lys Arg 115 120 125 27 420 DNA Artificial Sequence NovelSequence 27 aagcttgccg ccaccatggg ctggagctgt atcatcctct tcttagtagcaacagctaca 60 ggtgtccact ccgaggtcca actggtagaa tctggaggtg gtctcgtacagccaggagga 120 tctctgcgac tgagttgcgc cgtctctggg ttatcattaa ctagtaatagtgtgaactgg 180 atacggcaag cacctggcaa gggtctcgag tgggttggac taatatggagtaatggagac 240 acagattata attcagctat caaatctcga ttcacaatct ctagagacacttcgaagagc 300 accgtatacc tgcagatgaa cagtctgaga gctgaagata ctgcagtctactactgtgct 360 cgtgagtact atggatattt cgactattgg ggtcaaggta ccctagtcacagtctcctca 420 28 135 PRT Artificial Sequence Novel Sequence 28 Met GlyTrp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly 1 5 10 15 ValHis Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln 20 25 30 ProGly Gly Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Leu Ser Leu 35 40 45 ThrSer Asn Ser Val Asn Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu 50 55 60 GluTrp Val Gly Leu Ile Trp Ser Asn Gly Asp Thr Asp Tyr Asn Ser 65 70 75 80Ala Ile Lys Ser Arg Phe Thr Ile Ser Arg Asp Thr Ser Lys Ser Thr 85 90 95Val Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr 100 105110 Tyr Cys Ala Arg Glu Tyr Tyr Gly Tyr Phe Asp Tyr Trp Gly Gln Gly 115120 125 Thr Leu Val Thr Val Ser Ser 130 135

1. A RAM having affinity for human IL-5 antigen and comprising antigenbinding regions derived from heavy and/or light chain variable domainsof a donor antibody having affinity for human IL-5, the RAM having abinding affinity similar to that of the donor antibody.
 2. The anti-IL-5antibody molecule of claim 1 comprising a composite heavy chain and acomplementary light chain, said composite heavy chain having a variabledomain comprising predominately acceptor antibody heavy chain frameworkresidues and donor antibody heavy chain antigen-binding residues, saiddonor antibody having affinity for human IL-5, wherein, said compositeheavy chain comprises donor residues at least at positions 31-35, 50-65and 95-102 (according to the Kabat numbering system).
 3. The antibodymolecule of claim 2, wherein, amino acid residues 24, 27-30, 37, 49, 73,76 and 78 in said composite heavy chain are additionally donor residues.4. The antibody molecule of claim 2, wherein amino acid residues 23, 24,27-30, 37, 49, 73 and 76-78 in said composite heavy chain areadditionally donor residues.
 5. The anti-IL-5 antibody molecule of claim1 comprising a composite light chain and a complementary heavy chain,said composite light chain having a variable domain comprisingpredominately acceptor antibody light chain framework residues and donorantibody light chain antigen-binding residues, said donor antibodyhaving affinity for human IL-5, wherein, said composite light chaincomprises donor residues at least at positions 24-34, 50-56 and 89-97(according to the Kabat numbering system) and wherein, said anti-IL-5antibody molecule has a binding affinity similar to that of the donorantibody.
 6. The antibody molecule of claim 5 wherein amino acidresidues 68 and 71 in said composite light chain are additionally donorresidues.
 7. The antibody molecule of claim 5 wherein amino acidresidues 22, 68 and 71 in said composite light chain are additionallydonor residues.
 8. An anti-IL-5 antibody molecule having affinity forhuman IL-5 compressing the composite heavy chain of any one of claims 2to 4 and the composite light chain of any one of claims 5 to
 7. 9. Theantibody molecule of any one of claims 2 to 8 comprising predominantlyhuman acceptor residues and non-human donor residues.
 10. The antibodymolecule of any one of claims 2 to 9 wherein the acceptor residues forthe composite heavy and light chains are human group III heavy chain andhuman group I light chain residues respectively, and the donor residuesfor the composite heavy and light chains are rat 39D10 heavy and lightchain residues respectively.
 11. A DNA sequence which encodes for thecomposite heavy chain or the composite light chain of an antibodyaccording to any one of claims 2 to
 10. 12. A cloning or expressionvector containing a DNA sequence according to claim
 11. 13. A host celltransformed with a DNA sequence according to claim
 11. 14. A process forthe production of an anti-IL-5 antibody comprising expressing at leastone DNA sequence according to claim 11 in a transformed host cell.
 15. Aprocess for producing an anti-IL-5 antibody molecule comprising: (a)producing in an expression vector an operon having a DNA sequence whichencodes a composite heavy chain according to any one of claims 2 to 4;(b) optionally producing in an expression vector an operon having a DNAsequence which encodes a complementary light chain which may be acomposite light chain according to any one of claims 5 to 7; (c)transvecting a host cell with the or each vector; and (d) culturing thetransvected cell line to produce the antibody product.
 16. A process forproducing an anti-IL-5 antibody molecule according to claim 15 whereinthe DNA sequences encode a composite light chain and a complementaryheavy chain, respectively.
 17. A therapeutic or diagnostic compositioncomprising the antibody molecule according to any one of claims 1 to 10in combination with a pharmaceutically acceptable carrier, diluent orexcipient.
 18. A method of therapy or diagnosis comprising administeringan effective amount of an antibody molecule according to any one ofclaims 1 to 10 to a human or animal subject.